www.scielo.br/jaos http://dx.doi.org/10.1590/1678-775720160044 In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

Talita Signoreti GRAZIANO1, Caroline Morini CALIL2, Adilson SARTORATTO3, Gilson César Nobre FRANCO4, Francisco Carlos GROPPO5, Karina COGO-MÜLLER6

1- Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Área de Microbiologia e Imunologia, Departamento de Diagnóstico Oral, Piracicaba, SP, Brasil. 2- Centro de Diagnóstico de Halitose – Halicenter, São Paulo, SP, Brasil. 3- Universidade Estadual de Campinas, Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Campinas, SP, Brasil. 4- Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Área de Farmacologia, Anestesiologia e Terapêutica, Departamento de Ciências Fisiológicas, Piracicaba, SP, Brasil. 8QLYHUVLGDGH(VWDGXDOGH3RQWD*URVVD'HSDUWDPHQWRGH%LRORJLD*HUDO/DERUDWyULRGH)LVLRORJLDH3DWR¿VLRORJLD3RQWD*URVVD35%UDVLO 6- Universidade Estadual de Campinas, Faculdade de Ciências Farmacêuticas, Campinas, SP, Brasil.

Corresponding address:.DULQD&RJR0OOHU5XD6pUJLR%XDUTXHGH+RODQGD&%,,VDOD(ž3LVR&DPSLQDV63%UD]LOHPDLO [email protected]

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ABSTRACT

bjective: Halitosis can be caused by microorganisms that produce volatile sulphur Ocompounds (VSCs), which colonize the surface of the tongue and subgingival sites. Studies have reported that the use of natural products can reduce the bacterial load and, consequently, the development of halitosis. The aim of this study was to evaluate the antimicrobial activity of the essential oil of Melaleuca alternifolia on the growth and volatile sulphur compound (VSC) production of oral bacteria compared with chlorhexidine. Material and Methods: The effects of these substances were evaluated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in planktonic cultures of Porphyromonas gingivalis and Porphyromonas endodontalis. In addition, gas chromatography analyses were performed to measure the concentration of VSCs from bacterial cultures and to characterize M. alternifolia oil components. Results: The MIC and MBC values were as follows: M. alternifolia - P. gingivalis (MIC and MBC=0.007%), P. endodontalis (MIC and MBC=0.007%=0.5%); chlorhexidine - P. gingivalis and P. endodontalis (MIC and MBC=1.5 Pg/mL). M. alternifoliaVLJQL¿FDQWO\UHGXFHGWKHJURZWK

DQGSURGXFWLRQRIK\GURJHQVXO¿GH +2S) by P. gingivalis (p<0.05, ANOVA-Dunnet) and the

H2S and methyl mercaptan (CH3SH) levels of P. endodontalis (p<0.05, ANOVA-Dunnet). Chlorhexidine reduced the growth of both microorganisms without altering the production

of VSC in P. endodontalis. For P. gingivalis, the production of H2S and CH3SH decreased (p<0.05, ANOVA-Dunnet). Conclusion: M. alternifolia can reduce bacterial growth and VSCs production and could be used as an alternative to chlorhexidine.

Keywords: Products with antimicrobial action. Halitosis. Natural products. Porphyromonas gingivalis. Porphyromonas endodontalis.

INTRODUCTION be diagnosed and treated1,14,21. This condition is multifactorial and may comprise both oral and non- Halitosis, also known as bad breath or malodour, oral causes21,25. Periodontal disease, peri-implantitis, is a condition caused by fetid odours present in air deep carious lesions, tongue coating, impacted food emanating from the mouth1, leading to personal or debris, unclean dentures, and other oral problems and social discomfort21. The origin of pathological may contribute to the onset of halitosis9. halitosis can be systemic or local14 and should Oral bad breath can result from the degradation of

J Appl Oral Sci. 582 2016;24(6):582-9 In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria proteins containing sulphur amino acids (methionine chromatograph, model: HP-6890 (HP; Palo Alto, and cysteine), resulting in the production of California, USA), interfaced with a mass selective volatile sulphur compounds (VSCs), represented detector HP-5975. A fused silica capillary column by hydrogen sulfide (H2S), methyl mercaptan HP-5 (length of 30 m, internal diameter of 0.25

(CH3SH), and dimethyl sulphide [(CH3)2S]: gases PPDQG¿OPWKLFNQHVVRIP— ZDVXVHGZLWK that emanate malodour1. Some anaerobic gram- helium as the carrier gas (1 mL min-1). negative bacteria present in the oral cavity, such The oil was diluted with ethyl acetate, and 0.1 as Porphyromonas gingivalis, Fusobacterium mL was injected into the device. The temperatures nucleatum, Prevotella intermedia, Tannerella used were 220°C for the injector, 250°C for the forsythia, and Porphyromonas endodontalis, are detector, and 60°C – 240°C for the column (3°C min- the main species responsible for the production of 1). To identify the analytes, a mixture of n-alkanes VSCs18. In addition to the role of VSCs in generating was used to calculate the retention index (RI). halitosis, there is evidence suggesting that these Comparisons were performed using the National gases are also involved in the pathogenesis of Institute of Standards and Technology (NIST) periodontal diseases1,29. electronic library and literature data based on RI. Various oral approaches have been employed to The determination of essential oil components was treat halitosis, including the mechanical removal based on the calculation of the area under the peaks. RI WRQJXH DQG VXEJLQJLYDO ELR¿OPV WKH XVH RI chlorhexidine, cetylpyridinium, or essential oil Bacterial strains and culture conditions mouthrinses, and the application of masking P. gingivalis W83 and P. endodontalis (isolated products such as chewing gums and mouthrinses from clinical sample) were cultivated in Tryptic containing chlorine dioxide and zinc salts5,10,23,26. It Soy Broth (TSB – Difco Co.; Detroit, Michigan, has also been reported that natural products, such USA) or TSA (Tryptic Soy Agar - Difco Co.; Detroit, as green tea, produce effects that control halitosis Michigan, USA), both supplemented with hemin (5 and VSC production12,28,30. Most products used to μg/mL), menadione (1 μg/mL), and 2% of Yeast reduce malodour have antimicrobial properties, Extract (Difco Co.; Detroit, Michigan, USA). Growth and the decrease in VSCs is usually related to the and cultivation were performed under anaerobic suppression of bacterial growth. conditions (10% CO2, 10% H2, and 80% N2) using an Melaleuca alternifolia, also known as tea tree anaerobic chamber (MiniMacs Anaerobic Workstation oil, has been studied because of its antimicrobial  'RQ :KLWOH\ 6FLHQWL¿F 6KLSOH\ :HVW tea tree oil was shown to reduce the levels of malodour Minimum inhibitory concentration (MIC) and and production of VSCs in patients nursed in an minimum bactericidal concentration (MBC) intensive care unit10. Despite the antimicrobial MIC and MBC were carried out according to potential of M. alternifolia, there are few studies Clinical and Laboratory Standards Institute (CLSI)5 evaluating its activity against oral pathogens that ZLWK VRPH PRGL¿FDWLRQV )RU WKH GHWHUPLQDWLRQ cause bad breath4,8. Thus, the aim of this study of MIC and MBC, serial two-fold dilutions were was to evaluate the effects of Melaleuca alternifolia made for all substances tested. Concentrations oil and chlorhexidine on the viability and VSC ranged from 0.5% (v/v) to 0.002% (v/v) for the production of P. gingivalis and P. endodontalis. essential oil of M. alternifolia and from 100 μg/mL (0.01% – p/v) to 0.38 μg/mL (0.0038% – p/v) for MATERIAL AND METHODS the chlorhexidine solution. An inoculum of 40% transmittance, equivalent to approximately 8x108 Substances tested CFU/mL, was prepared from bacterial cultures on This study used the essential oil of Melaleuca TSA with three days of growth, and 500 μL samples alternifolia (Arista Industries; Wilton, Connecticut, of this bacterial suspension were transferred to USA) as the tested substance and chlorhexidine WXEHV FRQWDLQLQJ WKH WHVW VXEVWDQFHV LQ D ¿QDO gluconate (Sigma-Aldrich; St. Louis, Missouri, USA) volume of 6 mL. Moreover, tubes without tested as the standard antimicrobial. compounds or bacterial suspension were used as controls. Cultures were maintained under anaerobic Determination of the chemical profile conditions for 48 hours. The lowest concentration of M. alternifolia essential oil by gas of each substance with no bacterial growth was chromatography-mass spectrometry (GC- considered the MIC. For MBC determination, 10 MS) μL samples from TSB cultures were transferred The essential oil was subjected to gas WR 76$ SODWHV DQG LQFXEDWHG IRU ¿YH GD\V XQGHU chromatography analyses to obtain its chemical anaerobic conditions. The lowest concentration with profile. Analyses were performed on a gas no bacterial growth was considered the MBC.

J Appl Oral Sci. 583 2016;24(6):582-9 GRAZIANO TS, CALIL CM, SARTORATTO A, FRANCO GCN, GROPPO FC, COGO-MÜLLER K

In vitro production of Volatile Sulphur Chuo-ku, Osaka, Japan). After measurement, the Compounds (VSCs) device provided the concentrations of hydrogen

Sub-MIC concentrations of M. alternifolia oil and sulfite (H2S), methylmercaptan (CH3SH), and chlorhexidine were tested to evaluate their effects GLPHWK\OVXO¿GH> &+3)2S] in parts per billion. The on VSC production and to ensure bacterial growth tubes were also subjected to absorbance readings and gas production. Thus, concentrations tested NJ QP LQD8QLFR56VSHFWURSKRWRPHWHU were as follows: 0.002%, 0.001%, and 0.0005% (Unico Inc; Dayton, New Jersey, USA). for M. alternifolia; 0.38 μg/mL (0.0038%), 0.19 μg/mL (0.0019%), 0.095 μg/mL (0.00095%), Statistical analysis and 0.048 μg/mL (0.00048%) for chlorhexidine All experiments were performed in eight digluconate; representing concentrations 4, 8, and replicates and reproduced at least two times. The 16 times smaller than the MIC (4x

Table 1-,GHQWL¿FDWLRQRIDQDO\WHVRIM. alternifolia essential oil compared with the standard composition recommended by ISO 4730; a) TR – peak retention time (in minutes); b) fraction in percent of total integrated area for the chromatogram

(a) (b) tR (min) $QDO\WHVLGHQWL¿HG Relative % % recommended (ISO 4730:2004) 4.66 alpha-thujene 0.72 --- 4.84 alpha-pinene 1.98 1.0-6.0 5.87 beta-pinene 0.51 --- 6.23 beta-myrcene 0.59 --- 6.63 alpha-phellandrene 0.35 --- 7.06 alpha-terpinene 9.13 0.5-13 7.27 p-cymene 2.49 0.5-12 7.47 1,8-cineole (Eucalyptol) 3.42 ” 8.5 gamma-terpinene 19.72 10-28 9.41 terpinolene 3.03 1.5-5.0 13.2 terpinen-4-ol 42.07 • 13.52 alpha-terpineol 2.88 1.5-8.0 22.19 alpha-gurjunene 0.39 --- 22.58 trans-caryophyllene 0.36 --- 23.39 aromadendrene 1.33 Trace-7.0 24.24 alloaromadendrene 0.58 --- 25.48 cis-beta-guaiene 0.17 --- 25.85 alpha-muurolene 0.14 --- 26.79 delta-cadinene 1.61 Trace-8.0 27.09 cadina-1,4-diene 0.19 --- 29.33 YLULGLÀRURO 0.18 Trace- 1.5 29.43 cubeban-11-ol 0.16 --- 30.7 1-epi-cubenol 0.21 --- 59.58 squalene 5.24 ---

J Appl Oral Sci. 584 2016;24(6):582-9 In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

RESULTS In vitro production of Volatile Sulphur Compounds Chemical pro¿le of the essential oil of M. The effects of sub-MIC concentrations of tea tree alternifolia oil and chlorhexidine on the growth and production

The characterization of essential oil compounds of volatile sulphur gases (H2S and CH3SH) are shown was performed by comparing retention times and LQ ¿JXUHV  DQG  IRU P. gingivalis W83, and in MS/MS mass spectra of each peak with information Figures 3 and 4 for P. endodontalis. Concentrations LQWKH1,67OLEUDU\7KHFRPSRXQGVLGHQWL¿HGDQG tested were lower than the minimum inhibitory the percentage range for tea tree oil recommended concentration (sub-MIC concentrations) and were by ISO 4730:2004 are described in Table 1. GH¿QHGLQSUHYLRXVWHVWV The tea tree oil reduced the growth of P. gingivalis Minimum Inhibitory Concentration (MIC) : DQG VLJQL¿FDQWO\ FKDQJHG WKH SURGXFWLRQ RI and Minimum Bactericidal Concentration CH3SH at all concentrations tested (16x

The MIC and MBC values for M. alternifolia oil concentration promoted less CH3SH production than and chlorhexidine solution are shown in Table 2. The higher concentrations (p<0.05). The production of tea tree oil and chlorhexidine digluconate solution H2S was not altered by the essential oil at any of the were able to inhibit the growth of both strains at concentrations. In the presence of chlorhexidine, low concentrations. P. gingivalis W83 showed a reduction in growth at concentrations of 8x

Furthermore, it exhibited a reduction in CH3SH

levels for all concentrations tested and in H2S for Table 2- Values of MIC and MBC for M. alternifolia essential oil and for the chlorhexidine digluconate solution

Bacterial Strains M. alternifolia Chlorhexidine M. alternifolia Chlorhexidine P. gingivalis W83 0.007 % 1.5 μg/ml (0.00015 %) 0.007 % 1.5 μg/ml (0.00015 %) P. endodontalis 0.007 % 1.5 μg/ml (0.00015 %) 0.5 % 1.5 μg/ml (0.00015 %)

Figure 1- Effects of sub-MIC concentrations of tea tree oil on growth (D.O. – 660 nm) and production of volatile sulphur gases (H2S and CH3SH) for P. gingivalis : 6LJQL¿FDQW GLIIHUHQFHV DPRQJ WUHDWPHQWV DQG WKH FRQWURO JURXS ZHUH considered when p<0.05 (ANOVA, Tukey test). Different letters represent differences among groups

J Appl Oral Sci. 585 2016;24(6):582-9 GRAZIANO TS, CALIL CM, SARTORATTO A, FRANCO GCN, GROPPO FC, COGO-MÜLLER K

Figure 2- Effects of sub-MIC concentrations of chlorhexidine on growth (D.O. – 660 nm) and production of volatile sulphur gases (H2S and CH3SH) for P. gingivalis : 6LJQL¿FDQW GLIIHUHQFHV DPRQJ WUHDWPHQWV DQG WKH FRQWURO JURXS ZHUH considered when p<0.05 (ANOVA, Tukey test). Different letters represent differences among groups

Figure 3- Effects of sub-MIC concentrations of tea tree oil on growth (D.O. – 660 nm) and production of volatile sulphur gases (H2S and CH3SH) for P. endodontalis 6LJQL¿FDQW GLIIHUHQFHV DPRQJ WUHDWPHQWV DQG WKH FRQWURO JURXS ZHUH considered when p<0.05 (ANOVA, Tukey test). Different letters represent differences among groups

J Appl Oral Sci. 586 2016;24(6):582-9 In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

Figure 4- Effects of sub-MIC concentrations of chlorhexidine on growth (D.O. – 660 nm) and production of volatile sulphur gases (H2S and CH3SH) for P. endodontalis6LJQL¿FDQWGLIIHUHQFHVDPRQJWUHDWPHQWVDQGWKHFRQWUROJURXSZHUH considered when p<0.05 (ANOVA, Tukey test). Different letters represent differences among groups

8x

J Appl Oral Sci. 587 2016;24(6):582-9 GRAZIANO TS, CALIL CM, SARTORATTO A, FRANCO GCN, GROPPO FC, COGO-MÜLLER K values differed between studies, MBC values were and P. endodontalis, reducing the growth and the similar. Differences in MIC values may be due to production of VSCs at sub-MIC concentrations, different strains tested: in this study we used the comparably to chlorhexidine. Future studies can W83 strain, while Takarada, et al.22 (2004) used be conducted focusing on the development of the ATCC 33277, 53977, Su63, and W50 strains. pharmaceutical products containing M. alternifolia To the best of our knowledge, there are no previous oil for halitosis treatment. studies in the literature showing the effects of M. alternifolia on P. endodontalis. ACKNOWLEDGEMENTS To evaluate the activity of M. alternifolia and chlorhexidine on the production of volatile sulphur This study was supported by grants from FAPESP compounds, sub-inhibitory concentrations of these – São Paulo Research Foundation (2009/06037- substances were used. The sub-MIC concentrations 8 and 2009/14736-3). of the tea tree oil affected the growth of both microorganisms, reducing the growth at higher REFERENCES concentrations (8x<, 4xantiseptic TV. Susceptibility of oral bacteria to Melaleuca alternifolia (tea mouth rinse10,QDGGLWLRQĮWHUSLQHRORQHRIWKH tree) oil in vitro. Oral Microbiol Immunol. 2003;18(6):389-92. 9- Hinode D, Fukui M, Yokoyama N, Yokoyama M, Yoshioka FRPSRXQGV SUHVHQW LQ WHD WUHH RLO DQG LGHQWL¿HG M, Nakamura R. Relationship between tongue coating and in our study, has demonstrated activity against a secretory-immunoglobulin A level in saliva obtained from patients number of oral pathogens involved in periodontal complaining of oral malodor. J Clin Periodontol. 2003;30(12):1017- disease and caries17. 23. Chlorhexidine is widely used in mouthrinses, 10- Hur MH, Park J, Maddock-Jennings W, Kim DO, Lee MS. Reduction of mouth malodour and volatile sulphur compounds in causing membrane disruption and inhibition of intensive care patients using an essential oil mouthwash. Phytother proteolytic and glycosidic enzimes, leading to Res. 2007;21(7):641-3. growth inhibition and cell death27. Despite being 11- Karbach J, Ebenezer S, Warnke PH, Behrens E, Al-Nawas a potent antimicrobial, it has certain side effects B. Antimicrobial effect of Australian antibacterial essential oils such as altered taste, mucosal desquamation, tooth as alternative to common antiseptic solutions against clinically relevant oral pathogens. Clin Lab. 2015;61(1-2):61-8. VWDLQLQJLQFUHDVHGFDOFL¿HGVXSUDJLQJLYDOGHSRVLWV 12- Lodhia P, Yaegaki K, Khakbaznejad A, Imai T, Sato T, Tanaka 19 and a burning sensation in the oral mucosa . T, et al. Effect of green tea on volatile sulfur compounds in mouth Compared with chlorhexidine, tea tree oil showed air. J Nutr Sci Vitaminol (Tokyo). 2008;54(1):89-94. similar antimicrobial activity, promoting bactericidal 13- Loughlin R, Gilmore BF, McCarron PA Tunney MM. Comparison and bacteriostatic effects at low concentrations. of the cidal activity of tea tree oil and terpinen-4-ol against FOLQLFDOEDFWHULDOVNLQLVRODWHVDQGKXPDQ¿EUREODVWFHOOV/HWW$SSO Thus, tea tree oil could be a good alternative to Microbiol. 2008;46(4):428-33. chlorhexidine in oral hygiene products, mainly 14- Luqman M. Systemic origin of halitosis : a review. Int J Clin mouthrinses. Therefore, the development of new Dent Sci. 2012;3(1):15-9. mouthrinses containing tea tree oil and clinical 15- Makino Y, Yamaga T, Yoshihara A, Nohno K, Miyazaki H. studies testing these products are necessary. Association between volatile sulfur compounds and periodontal disease progression in elderly non-smokers. J Periodontol. In conclusion, M. alternifolia oil showed 2012;83(5):635-43. antimicrobial activity against P. gingivalis W83

J Appl Oral Sci. 588 2016;24(6):582-9 In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

16- Nakano Y, Yoshimura M, Koga T. Correlation between oral 24- Thosar N, Basak S, Bahadure RN, Rajurkar M. Antimicrobial malodor and periodontal bacteria. Microbes Infect. 2002;4(6):679- HI¿FDF\RI¿YHHVVHQWLDORLOVDJDLQVWRUDOSDWKRJHQVDQin vitro 83. study. Eur J Dent. 2013;7(1):1-7. 17- Park SN, Lim YK, Freire MO, Cho E, Jin D, Kook JK. Antimicrobial 25- Van den Broek A, Feenstra L, de Baat C. A review of the current HIIHFW RI OLQDORRO DQG ĮWHUSLQHRO DJDLQVW SHULRGRQWRSDWKLF DQG literature on management of halitosis. Oral Dis. 2008;14(1):30-9. cariogenic bacteria. Anaerobe. 2012;18(3):369-72. 26- Van Leeuwen MP, Slot DE, Van der Weijden GA. Essential oils 18- Persson S, Claesson R Carlsson J. The capacity of subgingival compared to chlorhexidine with respect to plaque and parameters microbiotas to produce volatile sulfur compounds in human serum. RI JLQJLYDO LQÀDPPDWLRQ D V\VWHPDWLF UHYLHZ - 3HULRGRQWRO Oral Microbiol Immunol. 1989;4(3):169-72. 2011;82(2):174-94. 19- Prayitno S, Addy M. An in vitro study of factors affecting the 27- Van Leeuwen MP, Slot DE, Van der Weijden GA. The effect of development of staining associated with the use of chlorhexidine. an essential-oils mouthrinse as compared to a vehicle solution J Periodontal Res. 1979;14(5):397-402. RQ SODTXH DQG JLQJLYDO LQÀDPPDWLRQ D V\VWHPDWLF UHYLHZ DQG 20- Sciarrone D, Ragonese C, Carnovale C, Piperno A, Dugo P, Dugo meta-analysis. Int J Dent Hyg. 2014;12(3):160-7. G, et al. Evaluation of tea tree oil quality and ascaridole: a deep 28- Xu X, Zhou XD, Wu CD. Tea catechin EGCg suppresses the mgl study by means of chiral and multi heart-cuts multidimensional gas gene associated with halitosis. J Dent Res. 2010;89(11):1304-8. chromatography system coupled to mass spectrometry detection. 29- Yaegaki K, Qian W, Murata T, Imai T, Sato T, Tanaka T, et J Chromatogr A. 2010;1217(41):6422-7. al. Oral malodorous compound causes apoptosis and genomic 21- Seemann R, Duarte da Conceição M, Filippi A, Greenman '1$ GDPDJH LQ KXPDQ JLQJLYDO ¿EUREODVWV - 3HULRGRQWDO 5HV J, Lenton P, Nachnani S, et al. Halitosis management by the 2008;43(4):391-9. general dental practitioner - results of an International Consensus 30- Zeng QC, Wu AZ, Pika J. The effect of green tea extract on Workshop. Swiss Dent J. 2014;124(11):1205-11. the removal of sulfur-containing oral malodor volatiles in vitro 22- Takarada K, Kimizuka R, Takahashi N, Honma K, Okuda K, and its potential application in chewing gum. J Breath Res. .DWR7$FRPSDULVRQRIWKHDQWLEDFWHULDOHI¿FDFLHVRIHVVHQWLDORLOV 2010;4(3):036005. against oral pathogens. Oral Microbiol Immunol. 2004;19(1):61-4. 23- Thaweboon S, Thaweboon B. Effect of an essential oil- containing mouth rinse on VSC-producing bacteria on the tongue. Southeast Asian J Trop Med Public Heal. 2011;42(2):456-62.

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